Impact of dendritic inhibition on neuronal excitation and plasticity
Final Report Abstract
The aim of the present research project was to gain a deeper understanding of the interplay of excitatory and inhibitory signaling in pyramidal neurons of the hippocampus. By taking advantage of a novel technique called two-color, two-photon uncaging of the excitatory neurotransmitter glutamate and the inhibitory neurotransmitter GABA, the local integration of these opposing signals in the fine processes of neurons was proposed to be studied. Unexpectedly, we found that slow hydrolysis of the caged GABA compound hindered detection of small current signals. While this finding forced us to deviate from the specific aims of the research proposal, we successfully completed four projects relevant to the general scope of the research project and beyond. (i) Two-color, one-photon uncaging of glutamate and GABA: Simultaneous optical activation of two different neurotransmitter receptors offers a unique means to study the underlying signal integration. To enable this with one-photon light, we developed a new long wavelength caged GABA (DEAC454-GABA) that has minimal absorption at shorter wavelength where traditional caging chromophores exhibit maximal absorption. By combining DEAC454-GABA with the short wavelength caged glutamate dcPNPP-glutamate, we were able to selectively stimulate glutamate and GABA receptors with two independent wavelengths of one-photon light. This allowed bidirectional modulation of the excitability of neurons in mouse brain slices. (ii) Comparative one- and two-photon uncaging of MNI-glutamate and MNI-kainate: Most caged glutamate and GABA compounds act as antagonists at GABAA receptors. In this study, we asked whether using the higher affinity agonist kainate might enable applying the caged compound at lower and therefore less antagonistic concentrations. Unexpectedly, we found that uncaging of MNI-kainate with different one- and two-photon light sources evoked smaller glutamatergic currents in hippocampal CA1 neurons compared to MNI-glutamate. While MNI-kainate is therefore not a suitable alternative to reduce GABAA receptor antagonism, we demonstrated for the first time two-photon uncaging of kainate at individual spines. This might allow studying the distribution of kainate receptors at high resolution by functional mapping. (iii) Thermodynamically stable, photoreversible pharmacology in neurons with one- and two-photon excitation: A common limitation of many current photoswitch molecules is their need to use cytotoxic UV light and their thermodynamic instability requiring constant illumination. To overcome these drawbacks, we developed the photoswitch 4FAB-QA. 4FAB-QA allowed bidirectional block of voltage-gated ion channels in neurons using visible light. Furthermore, the photoswitch could be preset to either isomer and used over several days for physiological experiments demonstrating its remarkable thermodynamic stability. Intriguingly, switching could also be effected with two-photon excitation significantly improving the spatial resolution of photoswitching. The 4FAB switch is therefore ideally suited for long-term physiological experiments in vitro and in vivo. (iv) Optical probing of acetylcholine receptors on neurons in the medial habenula with a novel caged nicotine drug analogue: Due to its chemical structure, the neurotransmitter acetylcholine cannot be attached to a caging chromophore. To circumvent this limitation, we caged the drug ABT594 which activates α4β3-subunit containing nicotinic acetylcholine receptors (nAChRs) with high affinity. ABT594 uncaging with oneand two-photon light enabled the fast activation of nAChRs on neurons of the medial habenula (MHb) known to express high levels of nAChRs. Functional mapping of receptor densities revealed expression of highly Ca2+-permeable nAChRs on the soma and dendrites of MHb neurons. Intriguingly, these receptors were also found on axons of MHb neurons where uncaging elicited considerable local increases in Ca2+ concentration. The unexpected presence of nAChRs on axons of MHb neurons might hint to a new signaling mechanism in this brain region. Furthermore, this finding is particularly interesting considering the role of the MHb in nicotine addiction. Our new probe will enable future studies to investigate the biology of nAChRs in diverse biological contexts. In the course of this research project, we were able to develop and apply several new optical tools for neurophysiology that will enable future studies to investigate neuronal excitability in health and disease.
Publications
- (2017). Two-color, one-photon uncaging of glutamate and GABA. PLOS ONE 12(11):e0187732
Passlick S, Kramer PF, Richers MT, Williams JT, Ellis-Davies GCR
(See online at https://doi.org/10.1371/journal.pone.0187732) - (2018). Comparative one- and two-photon uncaging of MNI-glutamate and MNI-kainate on hippocampal CA1 neurons. J Neurosci Methods 293:321–328
Passlick S, Ellis-Davies GCR
(See online at https://doi.org/10.1016/j.jneumeth.2017.10.013) - (2018). Optical probing of acetylcholine receptors on neurons in the medial habenula with a novel caged nicotine drug analogue. J Physiol
Passlick S, Thapaliya ER, Chen Z, Richers MT, Ellis-Davies GCR
(See online at https://doi.org/10.1113/JP276615) - (2018). Thermodynamically Stable, Photoreversible Pharmacology in Neurons with One- and Two-Photon Excitation. Angew Chem Int Ed Engl. 57:12554–12557
Passlick S, Richers MT, Ellis-Davies GCR
(See online at https://doi.org/10.1002/anie.201807880)
